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¹ Department of Pharmacology, University of Pittsburgh School of Medicine and ² Department of Medicine, University of Pittsburgh Cancer Institute, Pittsburgh, Pennsylvania

Requests for reprints: Daniel E. Johnson, Department of Medicine, University of Pittsburgh Cancer Institute, Hillman Cancer Center Research Pavilion, Room 2.18c, 5117 Centre Avenue, Pittsburgh, PA 15213-2582. Phone: 412-623-3245; Fax: 412-623-7768. E-mail: johnsond{at}pitt.edu

Treatment of cells with chemotherapy drugs activates the intrinsic mitochondrial pathway of apoptosis and the caspase protease cascade. Recently, the lysosomal protease cathepsin D has been implicated in apoptosis caused by oxidative stress, inhibition of protein kinase C, and stimulation of the TNFR1 and Fas death receptors. However, the role of cathepsin D in chemotherapy-induced cell death has remained largely unexplored. In this report, we show that treatment of U937 leukemia cells with the chemotherapy drug etoposide (VP-16) results in cathepsin D release into the cytosol within 4 hours after initiation of drug treatment. VP-16-induced cathepsin D release was not inhibited by z-VAD-FMK or pepstatin A, suggesting that it occurred independently of the activities of caspase proteases or cathepsin D. Down-regulation of cathepsin D expression in suspension U937 cells or adherent HeLa cells using cathepsin D small interfering RNA partially inhibited cell death resulting from treatment of cells with tumor necrosis factor-, tumor necrosis factor–related apoptosis inducing ligand, or the chemotherapy drugs VP-16, cisplatin, and 5-fluorouracil. Moreover, cathepsin D down-regulation significantly delayed cytochrome c release and caspase-3 activation in response to chemotherapy treatment. Incubation of isolated mitochondria with cathepsin D–treated cytosolic extracts resulted in potent release of cytochrome c, indicating that a cytoplasmic substrate mediates the effects of cathepsin D on mitochondria. Together, these findings show that cathepsin D plays an important role in chemotherapy-induced cell death, and that cathepsin D lies upstream of cytochrome c release and caspase-3 activation in the chemotherapy-induced execution pathway.

Key Words: cathepsin D • caspase-3 • chemotherapy • apoptosis • lysosome

Grant support: NIH grant RO1 CA86980 (to D.E. Johnson).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 11/ 9/04; revised 3/ 2/05; accepted 3/ 9/05.

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